Use of cross-protection to identify novel vaccine candidates for infectious agents

ABSTRACT

This invention discloses methods for identifying  Francisella tularensis  vaccine candidates. It enables identification of novel vaccine candidates and quality assurance for vaccine batches, assessment of protection in vaccinates and identification of the infecting agent in vaccinates. Mice were first vaccinated with  Brucella abortus  O-polysaccharide (OPS) vaccine. These animals were then given 10 LD 50 s of  F. tularensis  live vaccine strain (LVS). Sixty percent (60%) of the vaccinated mice survived the multiple lethal doses. Sera were collected from these surviving mice and the antibodies were used to probe supernatant and cell lysates of live  F. tularensis  LVS cultures. Several  F. tularensis  components were identified only by the noted “survivor” antisera. Of these identified proteins, enzyme digestions and chemical oxidation suggest post-translational modifications of some proteins e.g. a 52 kDa glycoprotein, a 45 kDa lipoprotein and a 19 kDa nucleoprotein. The 52 kDa component caused nitrous oxide induction in tissue cultures at low concentrations, cell death at high concentrations. Vaccination with this gave partial protection while addition of other components acted synergistically to give enhanced protection from 250 LD 50 s of  F. tularensis  LVS.

The present application is a divisional of Ser. No. 10/762,241, filedJan. 23, 2004 (allowed), which claims benefit of U.S. ProvisionalApplication No. 60/442,072, filed Jan. 24, 2003, the entire contents ofeach of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The general field of the invention is the development of sub-cellularvaccines that induce immunity to infectious agents. More particularly,the invention relates to the identification of novel vaccine candidates(with logical extensions to other infectious agents such as otherbacteria, fungi, yeast, viruses or parasites), quality assurance forvaccine batches, assessment of protection in vaccinated animals and theidentification of the infecting agent in vaccinates.

BACKGROUND OF THE INVENTION List of Prior Art Literature

-   Stewart, S. J. 1991. Francisella. In: Balows, A., W. J. Hausler,    Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.). Manual    of Clinical Microbiology. Am. Society for Microbiology, pp. 454-456.-   Franz, D. R., P. B. Jahrling, A. M. Friedlander, D. J.    McClain, D. L. Hoover, W. R. Bryne, J. A. Pavlin, G. W. Christopher    and E. M. Eitzen, Jr. 1997. Clinical recognition and management of    patients exposed to biological warfare agents. JAMA, 278:399-411.-   Evans, M. E., and A. M. Friedlander. Tularemia. In: F. R.    Sidell, E. T. Takafuji and D. R. Franz (ed.) Medical Aspects of    Chemical and Biological Warfare. 1997. Published by the Office of    the Surgeon General at TMM Publications. pp. 503-512.-   Cherwonogrodzky, J. W., M. H. Knodel, and M. R. Spence. 1994.    Increased encapsulation and virulence of Francisella tularensis live    vaccine strain (LVS) by subculturing on synthetic medium. Vaccine.    2:773-775.-   Corbel, M. J. Recent advances in the study of Brucella antigens and    their serological cross-reactions. 1985. Veterinary Bulletin. 55:    927-942.-   Sjostedt, A. 1997. Host-parasite interactions during tularemia.    (Introductory remarks to the presentation, given at the Medical    Protection B Conference, Munich, Germany).-   Golovliov, I., M. Ericsson, L. Akerblom, G. Sandstrom, A. Tarnvik    and A. Sjostedt. 1995. Adjuvanicity of ISCOMS incorporating a T-cell    reactive lipoprotein of the facultative intracellular pathogen    Francisella tularensis. Vaccine. 13:261-267.-   Hood, A. M. 1977. Virulence factors of Francisella tularensis. J.    Hyg. Camb. 79: 47-60-   Ancuta, P., T. Pedron, R. Girard, G. Sandstrom and R. Chaby. 1996.    Inability of the Francisella tularensis lipopolysaccharide to mimic    or to antagonize the induction of cell activation by endotoxins.    Infect. Immun. 64: 2041-2046.-   Conlan, J. W., H. Shen, A. Webb, M. B. Perry. 2002. Mice vaccinated    with the O-antigen of Francisella tularensis LVS lipopolysaccharide    conjugated to bovine serum albumin develop varying degrees of    protective immunity against systemic or aerosol challenge with    virulent type A and type B strains of the pathogen. Vaccine 20:    3465-3471.-   Cherwonogrodzky, J. W. 1983. Factors controlling haemolysin    production in Vibrio parahaemolyticus. Ph.D. thesis, University of    Toronto, pages 161-162.

Tularemia is primarily a disease of wildlife that spreads to humansincidentally such as by insect or tick bites, handling infectedcarcasses or by drinking contaminated water. The disease usuallyprogresses from an ulcer (at the site of infection or within the bowelif ingested) to oculoglandular infections (eyes are stressed and‘flu’-like symptoms such as chills, fever, headache and general achesand pains becoming progressively worse) then systemic gastrointestinalor pleuropneumonia tularemia that causes severe illness with a highmortality rate (30-60%) unless antibiotic therapy is given. Although theincidence of tularemia has declined with the decline of market huntingand trapping, it is still widespread around the globe, infectingwildlife, domestic animals and humans (Stewart, 1991). The bacterium isreadily grown on simple medium with a cysteine supplement, and it ishighly virulent when delivered as an aerosol or in contaminated water.It is a potential threat agent for biological warfare or terroristprograms (Franz et al., 1997).

With regards to medical countermeasures against tularemia, antibioticscan clear the infection, but the success of these antibiotics depends onwhere the infection has located and how early the patient is treated. AF. tularensis live vaccine strain (LVS) is available, but it has an IND(Investigational New Drug) status, its efficacy against exposures bydifferent routes of infection is questionable (Evans and Friedlander,1997) and under certain conditions it appears to revert to its virulentparental form (Cherwonogrodzky et al., 1994). There is therefore a needfor both a new, more effective and safe vaccine, a means of assessingthe stability of different batches of the existing LVS vaccine, and forassessing by a simple blood test if a vaccinate is indeed protected fromtularemia. As Brucella and F. tularensis cross-react (Corbel, 1985), itwould benefit serodiagnosis if an antigen could determine whichbacterium had infected a patient.

In theory, it might be viewed that sera from either vaccinated orinfected animals or humans might have antibodies that could identifycomponents key to the disease process and hence potentially useful asvaccine candidates. To date this has not happened. For the first part,vaccination often gives limited results (e.g. although the LVS protectsagainst tularemia, its efficacy for protecting against infection bydifferent routes is questionable) with antibody titers either being lowor rapidly diminishing with time. For the second part, infected animalsor human have an illness where the immunity has either failed or hasresponded incorrectly to what was required for protection. Indeed,Edward Francis (from whom Francisella is named) had 3 infections oftularemia and eventually died from this disease (Sjostedt, 1997). Aninfection did not provide Francis with immunity. Although sera fromvaccinated or infected patients do have antibodies with affinity for lowmolecular proteins of F. tularensis, these proteins do not appear togive protection when used as vaccines (Golovliov et al., 1995). Theapproaches used by the experts in the field to date teach away fromnovel approach of the present invention on how to identify potentiallyuseful components.

In a previous publication (Cherwonogrodzky et al., 1994), the capsule ofF. tularensis was viewed as a virulence factor. However, in subsequentstudies it was found that this was not the case. Mice did not become illwhen they were infected with F. tularensis live vaccine strainsubcultured more than 6 times in a synthetic salts medium, even thoughthese bacteria still had extensive capsules. Evidently the initialspeculation on the role of capsules for virulence of the bacterium wasincorrect and teaches away from the later findings.

With regards to toxins, the current state of knowledge is that toxinsare not present for F. tularensis (Hood, 1977; Ancuta et al., 1996). Inunpublished studies, we found that when the third (broth or agar)subculture had the supernatant filter-sterilized (0.2 μm filter used),and 0.05 ml was given to mice intranasally, initially the mice appearednormal. This initial observation appeared to confirm the existing stateof knowledge that no toxins were present in F. tularensis cultures.However, 24 hours after the supernatant inoculation, all mice were founddead in their cages. These latter (unreported) results proved, contraryto the literature, that a toxic agent was being produced by thebacterium but that it had a delayed action on the test animals.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a safe and effectivevaccine against tularemia. The methods disclosed will be applicable toother infectious diseases such as those caused by other bacteria, fungi,yeasts, viruses and parasites.

A mouse model is used which can be extended to any mammal. Mice arevaccinated with a polysaccharide from one bacterium (i.e. Brucella) soas to survive multiple lethal doses of another cross-reactive bacterium(F. tularensis). The “survivor” serum is used as a source of uniqueantibodies to identify virulence factors of the latter bacterium.

Specifically, mice were given a vaccine against one bacterium(O-polysaccharide, or OPS, vaccine which is protective against Brucellaabortus) but then infected with multiple lethal doses (10 LD₅₀) ofanother bacterium, Francisella tularensis. Due to cross-protection, mostof the mice vaccinated against brucellosis survived tularemia. It wasfound that these surviving vaccinated mice had in their sera antibodiesthat recognized the latter bacterial components expressed during thedisease process. That these mice had sera with antibodies thatrecognized previously overlooked proteins showed a novel method forfinding vaccine candidates and for assessing protection. That some ofthese components were specific to Francisella tularensis provided ameans of differentiating the infecting agent, even in the presence ofcross-reactions that currently confuse identification.

A further object of the present invention is to demonstrate theexpression of toxic components in culture supernatants that could beidentified and quantified with the novel antibodies. An application forthe assessment of these components is a quality assurance measure fordifferent vaccine lots of F. tularensis live vaccine strain.

According to one aspect of the invention, it provides a subcellularprotein expressed from Francisella tularensis infected mammal subculturegrowing in synthetic salts medium of weak acidity.

According to another aspect of the invention, it provides a method foridentifying an infectious agent in a mammal, comprising vaccinating themammal against a first infectious agent and subsequently exposing themammal to a second infectious agent to be identified, thereby causingthe mammal to express a subcellular protein against the secondinfectious agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 12% SDS PAGE, Silver Stain, of Francisella tularensislive vaccine strain (LVS) sequentially subcultured on synthetic saltsmedium (Chamberlain's) and the supernatants of either agar (A1-A6) orbroth (B1-B6) cultures inspected for proteins.

The far right lane is the Rainbow™ Marker (M) which has standardproteins of different molecular weights.

FIG. 2 shows a 12% SDS PAGE, Silver Stain, of Francisella tularensislive vaccine strain (LVS) subcultured on synthetic salts medium(Chamberlain's).

Lane A3S shows proteins released into the agar of agar subculture #1,B3S are the proteins released into the supernatant of broth subculture#3. Lane B3CL are the proteins from the cell lysate of broth subculture#3, A3CL are the proteins from the cell lysate of agar subculture #3.Lane M is the Rainbow™ molecular weight markers.

FIG. 3 shows an immunoblot (Western) of F. tularensis cellularcomponents.

Samples electrophoresed on an 12% SDS PAGE with 0.75 mm thickness andtransblotted as described. Primary antibody: 1:50 Brucella-OPS primedanti F. tularensis murine antisera. Secondary antibody: 1:3000 Caltaganti-mouse-HRP Lane 1: broth subculture 1, lane 2: broth subculture 2,lane 3: broth subculture 4, lane 4: broth subculture 5, lane 5: 5 μLRainbow MW marker.

FIG. 4 shows an immunoblot (Western) Characterization of F. tularensisLVS cellular components.

Samples prepared and electrophoresed under normal polarity on a 7.5%native (no SDS) gel of 1.0 mm thickness as described. Primary antibody:1:50 Brucella-OPS primed anti F. tularensis murine antisera. Secondaryantibody: 1:3000 Caltag anti-mouse-HRP Lane 1: BioRad H/L MW marker (5μL), Lane 2: 24 hr growth, Lane 3: 48 hr growth, Lane 4: 72 hr growth,Lane 5: 96 hr growth, Lane 6: 5 day growth, Lane 7: 6 day growth, Lane8: 7 day growth, Lane 9: 8 day growth.

FIG. 5 shows an elution profile (absorbance at 220 nm) of proteinseluted from a Pharmacia Mono-Q™ column.

Source of material was F. tularensis LVS lysate (cell disrupted bysonication) that was partially purified with an Amicon Filtration device(molecular weight cutoff of 30 kDa)

FIG. 6 indicates Nitric Oxide (NO) production by J774.1 macrophage cellsin response to treatment by Mono-Q separated fractions of F. tularensisLVS cell lysate for 24 hr.

FIG. 7 shows Vero tissue cultures.

Photograph on the left is the control; photograph on the right is celldeath as a result of the addition of 3 mg/ml of the F. tularensis LVS 52kDa protein.

FIG. 8 shows Nitrous oxide (NO) production of Vero tissue cultures whengiven increasing amounts of the F. tularensis 52 kDa protein.

FIG. 9 shows enzyme characterization and immunoblot (Western) ofantigenic F. tularensis liquid culture cellular components.

Supernatant from Day 7 of the multiday growth series used (7D).Supernatant concentrate (1.88 μg) was treated with various enzymes (0.83μg). Samples prepared and electrophoresed on a 7.5% SDS-PAGE of 11.0 mmthickness as described and transblotted onto nitrocellulose. Membraneprobed under the following conditions: Primary antibody: 1:1000Brucella-OPS primed anti F. tularensis murine antisera. Secondaryantibody: 1:3000 Caltag anti-mouse-HRP Lane 1: BioRad H/L MW marker (5μL), Lane 2: 7D supernatant, Lane 3: Proteinase-K digested supernatant,Lane 4: Proteinase-K, Lane 5: Lipase digested supernatant, Lane 6:Lipase, Lane 7: DNAse digested supernatant, Lane 8: DNAse, Lane 9: RNAsedigested supernatant, Lane 10: RNAse, Lane 11: Lysozyme digestedsupernatant, Lane 12: Lysozyme, Lane 13: BioRad Kailadoscope MW marker(6 μL).

DETAILED DESCRIPTION OF THE INVENTION

Our laboratory has taken an approach (i.e. identifying usefulsubcellular components) contrary to the existing scientific methods(i.e. forming live attenuated mutants) on tularemia vaccine development.Our successes were based on our past unreported observations:

-   -   (a) We have discovered that subculturing F. tularensis LVS in a        synthetic medium stresses the bacterium and causes enhanced        expression of a capsule. For other infectious bacteria (e.g.        those that cause whooping cough and pneumonia), the presence of        a capsule is indicative of the pathogenicity of the bacterium        and severity of illness in the host. This teaches away from our        later observation that, after a few subcultures in the synthetic        medium, bacteria continued to be capsulated but were non-lethal        for the mice tested. Thus, lethality is due to a factor or        factors other than the capsule.    -   (b) Toxins usually act immediately on host target cells because        of their detergent (e.g. bee venom toxin) or enzymatic (e.g.        diphtheria toxin) activity. The existing state of knowledge in        the scientific community is that F. tularensis does not produce        toxins. Our own work initially supported this accepted dogma.        When F. tularensis LVS was grown in synthetic media, cells        removed by centrifugation and then the culture supernatant        filter-sterilization (0.2 um filtration), no effects were        observed when 50 μl of this supernatant was inoculated into the        nostrils of anaesthetized mice. However on the second day, all        inoculated mice were found dead in their cages. There was an        unobvious toxin or toxins from F. tularensis that we almost        missed because these caused delayed death.    -   (c) In one study, F. tularensis LVS was reverted to a more        pathogenic state by subculturing in synthetic medium. Ten LD50s        were administered to several mice by different routes        (intra-nasally, intra-peritoneally, intra-venously). Past        results showed that mice died in 5-8 days when given this dose        and so on the fifth day mice were sacrificed to understand the        disease process leading to death. Curiously, despite the        different routes of administration of the bacterium and the        presence of the bacterium in the blood and organs, histology        revealed that only the lung was affected. For tularemia, it was        observed that microscopic foci of necrosis and pneumonia        occurred in the lungs of mice. The lack of significant histology        with the other tissues may give evidence why the effects of a        toxin have not been observed by other research laboratories.        Although we found that a toxin was produced, the action was        unobvious, affecting only susceptible tissue (lung) in        susceptible animals (mice). Had other tissues from other animals        been investigated, it is likely we would have also missed this        effect.

The usual method to identify a bacterial component is to vaccinate ananimal with the killed bacteria, take the serum and probe with antibodyin these components on acrylamide gels. Using commercial rabbitantiserum (rabbits that had been vaccinated with killed F. tularensiscells) did not reveal anything different from other reported studies.

It is known that Brucella abortus cross-reacts with F. tularensis. Micewere infected with live B. abortus (i.e. by using live untreated cellsthat would grow inside the host, it was hoped that key antigens would beintact for an antibody response), their serum collected,filter-sterilized and sterility-checked. However, this antiserum did notreveal anything different or striking when used to tag F. tularensiscell components separated on acrylamide gels.

Despite the above negative results that would have led anyone skilled inthe art to conclude that nothing different or striking was beingexpressed by the reverted F. tularensis, we believed otherwise. Ourunpublished results noted above showed that something toxic was beingexpressed but that standard methods for detection were inadequate. Toresolve this impasse, innovative methods were developed and used thatare described below.

Other investigators have used, as vaccines, killed or attenuated(weakened by genetically manipulation) microbial agents that do notexpress components critical to the disease process. Highly virulentagents are not used because the end result is a dead animal. Ourinvention used a vaccine to one bacterium (the O-polysaccharide fromBrucella abortus) to protect mice against several lethal doses (10LD₅₀s) of another bacterium, Francisella tularensis, bycross-protection. All mice initially did show preliminary symptoms ofinfection. About 60% of the vaccinated animals rapidly recovered fromthe multiple lethal doses. These mice had antibodies that identifiedpreviously unknown agent components. These components were notidentified with serum taken from mice infected with B. abortus orvaccinated with killed F. tularensis cells. Some of these componentswere characterized, purified and used as vaccines. These were also usedto assess the quality assurance for different vaccine (F. tularensisLVS) subcultures.

A recent publication (Conlan et al., 2002) teaches away from both theusefulness of B. abortus OPS vaccine to protect against tularemia, andto protect against a respiratory (i.e. intranasal) challenge oftularemia. For the former, the compositions of OPS from B. abortus andthat of F. tularensis are dissimilar. Whereas the OPS of B. abortus is a1,2 linked formamido-mannose polymer, the OPS of F. tularensis hasdifferent sugars and linkages. In addition, although the latter had someefficacy in protecting mice from intra-dermal challenges of F.tularensis, these “were completely unprotected against a low doseaerosol challenge with this strain”. In contrast, we did find that theB. abortus OPS vaccine protected mice from tularemia and from arespiratory challenge.

Another unobvious, and also unfortunate, observation was the effect offreeze-thaw on the special (“survivor”) serum. This caused theantibodies to bind to F. tularensis cell lysate proteins in a differentbinding profile. Results were similar to published information in theliterature whereby antibodies bound to a greater extent to low molecularweight components (shown by others to be of no use as vaccinecandidates) and far less to the larger molecular weight components. Asfrozen-and-thawed sera from control unvaccinated mice gave similarresults, it is likely that the freeze-thaw caused denaturation and hencenon-specific binding of the antibodies. Much of the work presented inthis patent, therefore, is based on identification of vaccine componentsby using fresh unfrozen serum, stored in the refrigerator at 4° C. whennot in use.

Materials and Methods Bacterial Cultures

Francisella tularensis live vaccine strain (LVS) was acquired as afreeze-dried vaccine in a vial (Lot #11, Code Number: NDBR 101, 2.4×10⁹cfu/ml when rehydrated) from the United States Army Medical ResearchInstitute of Infectious Diseases (USAMRIID), Frederick, Md., USA. It wasgrown either on agar or in broth synthetic medium at 37° C., 90%humidity and 5% CO₂. For intranasal inoculation, 10 LD₅₀ was 1×10⁵bacteria diluted in 37° C. prewarmed phosphate buffered saline (PBS).These bacteria were in 0.01 ml PBS given intranasally by Eppendorf™pipette tip to anaesthetized Balb/c mice. For intraperitonealinjections, 10 LD₅₀ was 1×10⁶ bacteria in 0.1 ml PBS.

Brucella abortus strain 30 was acquired from the Animal DiseasesResearch Insitute-Nepean, (ADRI-Nepean), Ontario. This was maintained onBrucella agar with 1.5 ppm crystal violet and incubated as for LVS. Thisculture was used to infect mice (within our Biocontainment Level 3facilities) for sera against live Brucella abortus.

Five-percent phenol-killed B. abortus 1119-3 was acquired from Dr. JanetPayeur, United States Department of Agriculture (USDA), Ames, Iowa.These cells were used as the source for O-polysaccharide vaccine whichwas purified by a method previously described (Cherwonogrodzky et al.,1990).

To compare antigen expression of LVS to other bacteria, 4 bacteria wereused. Staphylococcus aureus ATCC 25923 (as an antibody-binding positivecontrol) was from Fisher Scientific (Ottawa, Bactrol Discs Set A).Escherichia coli 0:157, H:7, Salmonella godesburg and Pseudomonasmaltophilia 555 (which all have OPS that cross-react with Brucella) wereobtained from Dr. Douglas Griffith (NRC, Ottawa).

Bacterial Growth Media

For F. tularensis LVS, the synthetic medium base consisted of 1% NaCl,0.4% glucose, 0.1% (each)/KH₂PO₄ and K₂HPO₄, 0.2% (each) L-proline andDL-threonine, 0.04% (each) of L-arginine, L-asparagine, DL-isoleucine,L-leucine, L-lysine HCl, DL-methionine, DL-serine, L-tyrosine andDL-valine, 0.02% (each) L-cystine HCl and L-histidine, 0.0135%MgSO₄.7H₂O, 0.40 parts per million (ppm) spermine phosphate, 4 ppmthiamine HCl, 2 ppm DL-calcium pantothenate and FeSO₄.7H₂O (in triplydistilled water, filter sterilized, pH 6.5). For broth cultures, a vialof stock culture was taken from the −70° C. freezer, partially thawedand a loopful was used to inoculate 10 ml peptone-cysteine broth tubes.These were incubated for a few days until growth was obvious. These werethen added to 500 ml synthetic medium base in 1 litre sterile flasksthen incubated for early logarithmic or late stationary phases (shakenat 150 rpm, 37° C.). For agar plates, a double strength of the syntheticmedium was made and placed in a 50° C. water bath, a 4% agar (DifcoLaboratories, Detroit, Mich.) in distilled water suspension wasautoclaved (30 min, 121° C., 15 psi), cooled to 50° C., then these werecombined and used to make pour plates.

For Brucella abortus 30, this was maintained on Brucella agar with 1.5ppm crystal violet, subcultured in Brucella broths (without crystalviolet) at 37° C., 5% CO₂ and 150 rpm.

For the 4 other bacteria noted, these were grown in Luria Broth (DifcoLaboratories, Detroit, Mich.; 10 g/L Bacto-peptone, 5.0 g/L Bacto yeastextract and 10 g/L sodium chloride), then incubated, centrifuged andsonicated as noted for LVS in this section.

Sonication

For sonicating particulate suspensions, a Soniprep 15™ and a ProcessTimer™ (both manufactured by MSE™) were used. Samples were in plastictubes which were held with a clamp and partially immersed in ice-waterin a small beaker. The amplitude of the 10 mm probe was adjustedmanually to 10 μm, the sequence was 5 cycles of 15 second pulsesfollowed by 1 min chilling. For the sonication of a live culture of F.tularensis LVS, two 500 ml cultures were centrifuged (10,000×g, 4° C.,30 min), the white pellet was resuspended in 75 ml of sterile PBS thenstored frozen at −70° C. Prior to use, this was thawed at roomtemperature (22° C.). Sonication on these freeze-thawed stressed cellswas as before except that 10 ml aliquots were used and 12 cycles wereused for a total homogenization time of 3 minutes. (Due to BL-2 aerosolhazards, the entire sonicator was placed inside a BioSafety Cabinet anddecontaminated with 70% ethanol from a spray bottle prior to removal.)The disrupted cell suspension was centrifuged (10,000×G, 30 min) thenthe supernatant of the cell lysate was saved.

Use of Animals (Mice)

Mice (usually balb/c, female, 19-21 g, 35 days old) were acquired fromCharles River (St. Constant, Quebec). Mice were cared for in accordancewith the guidelines set by the Canadian Council for Animal Care. Allprocedures were reviewed and approved by the DRDC Suffield Animal CareCommittee (members consist of a veterinarian, scientists and lay peoplefrom the community). Protocol JC-98-02 was used for this study. To raiseanti-tularemia mouse sera, twenty mice were first vaccinated with the B.abortus OPS. Each mouse was given 1 μg OPS in 0.1 ml sterile saline bythe intra-peritoneal route (i.p.). Two weeks later, these mice weretaken to a BioSafety Cabinet, infected with 10LD₅₀ of F. tularensis LVS(only BL-2 containment is required) which was diluted in sterileprewarmed PBS and given i.p. Infected mice were then placed intoisolator cages (with adequate food and water, with HEPA filter tops)which in turn were placed into a HEPA-filtered Animal Isolator (ThorenCaging, Toronto). The mice were monitored for 2 weeks. About 60% of theOPS vaccinated mice survived 10 LD₅₀ of F. tularensis LVS and then thesewere sacrified for their anti-tularemia serum. For infection by theintranasal route, mice were anaesthetized with 0.1 ml of 1:10 dilutedSomnitol™ given intraperitoneal (as anaesthetized mice are sensitive tothe cold, it is imperative that their surrounding temperature be about22° C., also a heat lamp at a distance may be needed to assist theirrecovery) then 0.01 ml of the bacterial suspension was delivered to anostril by an Eppendorf™ tip.

Sera

Blood was collected into 1.5 ml small Eppendorf™ centrifuge tubes. Smallvolumes (0.1-0.3 ml) were collected by inserting a 26-gauge needle intoa warmed tail vein (i.e. warmed with a heatlamp), then with anEppendorf™ tip transferring the blood from the needle cap to thecentrifuge tube. Larger volumes (1 ml) were collected by firstanaesthetizing mice with a double dose of Somnitol™, then doing aheart-puncture with a 1 ml syringe with a 26-gauge needle attached. Theblood was centrifuged (5,000×g, room temperature, 10 min) and the serumtransferred to another labeled centrifuge tube. Glycerol was added tothe serum for a final concentration of 10%. Most of the methods usedfresh unfrozen serum. It was later found that serum that was storedfrozen at −70° C. for extended times did not yield the same specificresults as when the serum was fresh and unfrozen.

Polyacrylamide Gel Electrophoresis (PAGE)

For resolution of lower molecular weight components, 12% polyacrylamidegels (reagents from Sigma Chemical Co., St. Louis, Mo.) were prepared bythe method of Laemnli (1970). For these, 1.88 ml of 40% acrylamide/1.3%BIS (N,N′methylene-bis-acrylamide), 1.45 ml 3 M TRIS(tris-hydroxymethyl-aminomethane), buffer, pH adjusted to 8.9 withhydrochloric acid, 0.5 ml 2% SDS (sodium dodecyl sulphate, do not usefor native gels), 10 μl TEMED (N,N,N′,N′-tetramethylethylenediamine),100 μl 10% ammonium persulphate and 6.08 ml of triple-distilled waterwere combined. For separating components of higher molecular weight, a7.5% polyacrylamide gel was used.

Electrophoresis was done in a BioRad™ minigel system following theprotocols outlined by the manufacturer. The acrylamide was loaded intothe glass chambers soon after preparation. About 10 ml was required for2 mini-gels. As noted below, water-saturated butanol was added to thetop of the liquid acrylamide to make an even surface. On top of theacrylamide was added the 4% “stacking gel”. This was made with 0.5 ml of40% acrylamide/1.3% BIS, 0.6 ml of 3 M TRIS (pH 8.9), 0.25 ml of 2% SDS(SDS is not used for native gels), 5 μl of TEMED, 50 μl of ammoniumpersulfate and 3.7 ml of triple-distilled water. The stacking gel wasloaded onto the acrylamide layer soon after preparation.

For electrophoresis, a 10-fold strength electrolyte running buffer wasmade by combining 30 g of TRIS base, 144 g glycine and 10 g SDS (do notuse for native gels) in 1 litre of triple distilled water. As only 500ml of buffer was needed for each run, 50 ml of this and 450 ml of waterwas combined.

A double-strength buffer for samples consisted of 100 mM TRIS-HCl (pH6.8) and 200 mM DTT (dithiothreitol) with 20% glycerol, 2% SDS (absentfor native gels) and 0.1% bromophenol blue.

For the procedure, 4 glass walls and 3 spacers were placed in theapparatus holder. Eight mm from the top of the glass walls was measured(one could use the stacking comb as a place holder), and then a line wasdrawn on the glass. With a 1 ml pipette and pipetter, the acrylamide (12or 7.5%) solution was added up to the marked line. Enoughwater-saturated butanol was added to provide a layer that evened thesurface of the acrylamide. When the acrylamide solidified, the butanolwas rinsed out with water, the water removed and the remaining excesswater removed by blotting with filter paper. A comb (thick 14 well comb)was inserted between the glass walls and stacking gel was dispensed overthe acrylamide. The stacking gel was allowed to set. The comb was thenremoved, the two gels were taken from the setting apparatus and thesewere placed in carriers to be placed in the mini-gel boat. For thebottom, the gel boat was filled with running buffer (single strength) to⅓ the height of the gels. For the top, running buffer was added and anybubbles in the stacking gel spaces were removed with a Pasteur pipette.A small amount of sample buffer was placed in each well to identify thelocation for sample loading. Samples were diluted 1:1 with 2-foldstrength sample buffer, boiled for 10 minutes, vortexed for 5 seconds,cooled, then centrifuged for 10 seconds at maximum speed in anEppendorf™ desktop centrifuge (244,000 rpm). A total of 10 μl wastransferred to each well in the gel. Samples were run at a constantvoltage of 100 V until the dye front reached the end of the gel.Duplicate gels were run concurrently for a protein stain and animmunoblot.

Protein Staining of Polyacrylamide Gels

Polyacrylamide gels were stained in large Petri dishes (from FisherScientific, Edmonton) containing 0.025% Coomassie Brilliant Blue R250,40% methanol, 7% acetic acid and about 53% water (this solution bothfixed and stained the proteins). The gels were placed in this solutionand the plate was rocked for a few hours. Destaining (removal of dyefrom the gel where proteins were not present) was done with severalchanges of 40% methanol, 7% acetic acid and 53% water. Once destained,the gels were stored in 5% glycerol in water.

Molecular weight standards (Bio-Rad Laboratories, Mississauga, Ontario),that were run as sample on the first and last wells, varied depending onthe desired range for the test samples. Broad range standards includedmyosin (200 kDa), beta-galactosidase (116 kDa), phosphorylase B (97kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonicanydrase (31 kDa), soybean trypsin inhibitor (22 kDa), lysozyme (14 kDa)and aprotinin (6.5 kDa). Low range standards ranged from phosphorylase Bto lysozyme, high range standards ranged from myosin to ovalbumin.

Western Immunoblots

Following electrophoresis, the gel was electro-blotted onto anitrocellulose membrane (Hybond, ECL Chemicals, Amersham Pharmacia LifeSciences, Baie Dorfe, Quebec) in a BioRad (Mississauga, Ontario) miniProtean-2-electrotransfer apparatus. The transfer buffer was 3 g ofTrizma-base (Sigma Chemicals, St. Louis, USA), 14.4 g glycine and 200 mlof methanol with water to make 1 litre. To block binding sites on thenitrocellulose membrane where proteins were not present, the membranewas placed into an 8 cm Petri dish containing a 5% solution of blockingreagent (ECL blocking agent, ECL Chemicals, Amersham Pharmacia LifeSciences, Baie Dorfe, Quebec) and phosphate buffered saline with 0.1%Tween-20 (PBS-Tween). This preparation was then incubated for an hour atroom temperature (22° C.) with gentle swirling on a flat-top rotationalshaker (set at 60 rpm). The membrane was then washed for 10 minutes inPBS-Tween, followed by 3 subsequent washes of 5 minutes each. Serum(from control unvaccinated mice, mice infected with B. abortus 30, micevaccinated then infected with 10 LD50 of F. tularensis LVS, or rabbitanti-F. tularensis antiserum) was the primary “detector” antibodydiluted 1:1000 in PBS-Tween. The nitrocellulose membrane was incubatedin 50 ml of this antibody solution for 1 hour in PBS-Tween as before.The second “indicator” antibody was a Caltag (Caltag Laboratories c/oCedarlane Laboratories Ltd., Homby, Ontario) anti-mouse (or anti-rabbit)antibody conjugated to horseradish peroxidase, diluted 1:30,000 in 50 mlof PBS-Tween and incubated at room temperature for an hour with gentleswirling. The membrane was then washed in PBS-Tween as noted above.Following the last wash, the membrane was placed onto a clean, dust-freesheet of acetate. A 5 ml volume of a 1:1 solution of A and B (ECLChemicals, Amersham Pharmacia Life Sciences, Baie Dorfe, Quebec) wasplaced onto the membrane and this activated the antibody-HRP tofluoresce and emit light. This was left for 1 minute on the membrane andthen the excess was wicked away with a paper towel. A second strip ofacetate was layered on top of the membrane (care was taken to avoidentrapped bubbles). This “sandwich” was then transferred into anexposure cassette and taped in place. Sensitized X-ray film (ECLHyperfilm, Amersham Pharmacia Life Sciences, Baie Dorfe, Quebec) wasadded to the cassette under safelight (red light) conditions.Fluorescent activated antibody-HRP was allowed to expose the film for 40minutes. The film was then removed from the cassette and developed inKodak GBX developer until it appeared dark by inspection under asafelight. The film was then fixed for 3 minutes and washed thoroughlyin distilled water for another 5 minutes. It was dried overnight,photographed and stored.

Silver Stain for Proteins and Lipopolysaccharide)

For proteins, the Bio-Rad™ (Mississauga, Ontario) silver stain was used(with dichromate as the oxidizer). For lipopolysaccharide (LPS),periodic acid was used to oxidize carbohydrates, such as those in theO-polysaccharide and core region of LPS, and to activate these for areaction with silver. The procedure of Bio-Rad™ silver staining forproteins was followed except that periodic acid was used, instead ofdichromate, as the oxidizer. The polyacrylamide gel was added to a Petridish and then 50 ml of “fixer” (25% isopropanol, 7% acetic acid, 68 mltriple distilled water) was added, incubated at room temperature for 15minutes, discarded, then fresh “fixer” was added and incubated foranother 15 minutes. The “fixer” was discarded, a few ml of “oxidizer”(0.7% periodic acid, 40% ethanol, 5% acetic acid, 54.3% water) was usedto rinse the plate, and then 50 ml of “oxidizer” was added and allowedto incubate for 5 minutes. The plate and gel were rinsed by adding 50 mlof distilled water and gently swirling this on a flat-top rotationalshaker for 10 minutes. The water was discarded and then 50 ml of “silverreagent” (the Bio-Rad™ silver staining solution was diluted 1:10 intriple distilled water) was added and the plate incubated for 20minutes. The gel was lightly rinsed for about 30 seconds with distilledwater delivered by a squeeze bottle and then “developer” (1.6% Bio-Rad™developer powder in water) was added (incubation was variable, anywherefrom 1-15 minutes depending on the reaction intensity). Fifty ml of 5%acetic acid was then added as the stopping bath.

Protein Determination

To conserve on test samples while being able to assess a wide range ofdilutions, a microtitre assay was done. Each assay was performed in a96-well, flat bottom, sterile microtitre plate (Nalgene™). Eightymicrolitres (80 μl) of triple-distilled water was added into wells 2through 12 for all rows (A-H) to be used. Eighty microlitres of thebovine serum albumin standard was added to well A1. Eighty microlitreswas also added to A2 (for 160 μl of a 1:2 dilution). Eighty microlitresfrom A2 was transferred to A3 and with unused pipette tips the standardwas diluted across the row. Test samples were diluted in the same mannerin the other rows. Eighty microlitres of the Bio-Rad™ Protein Assay Dyereagent concentrate (Bio-Rad Laboratories, Mississauga, Ontario) wasadded to each well, thoroughly mixed and then left to incubate for 5minutes at room temperature. The plate was then read at 595 nm in aThermomax™ microtitre plate reader (Molecular Devices, Sunnyvale,Calif.). The results were analyzed with the software program, Softmax™(also by Molecular Devices) using the supplied 4-parameter fit.

Sample Preparations

For Francisella tularensis LVS cultures grown in broths, after 3 days ofincubation the culture was centrifuged, then had the supernatant removedand filter-sterilized through a 0.22 μm Nalgene filter sterilizing flask(1 litre). Part of the supernatant volume was stored in therefrigerator, part was freeze-dried. The bacterial pellet wasresuspended in PBS, frozen at −70° C. then thawed when required andsonicated as noted in the previous section.

For cultures grown on agar plates, after incubation for about a week,cells were scraped off the agar surface, the agar from a plate was cutinto pieces, transferred to a plastic bag, 50 ml of sterile PBS wasadded, the bag sealed and then contents crushed on a Stomacher (Seward,UK) for 3 minutes. The bag was then opened, the slurry transferred to acentrifuge bottle, this was centrifuged (10,000×g, 4° C., 30 min), thesupernatant removed then filter-sterilized as before. Part of thislatter supernatant was stored in the refrigerator, part wasfreeze-dried.

Bacterial cell sonicates, with components released into PBS, wereseparated from unbroken cells or cell debris by centrifugation(10,000×g, 4° C., 30 min). The liquid (referred to as cell lysate) wasplaced into a Spectrapor 10,000 m.w. cutoff dialysis bag and dialyzed(overnight at 4° C.) against 1 mM HEPES and 1 mM DTT (dithiothreitol).

As required, the components of these various supernatant preparationswere concentrated with an Amicon ultrafiltration membrane (Millipore,Nepean, Ontario) with a 30,000 m.w. cutoff. Once the membrane was fittedinto the unit, and the preparations were added, the unit was sealed anddry nitrogen was used to pressurize the unit to 55 psi. The retentate,containing components larger than 30 kDa, was collected and stored at 4°C.

Nitric Oxide and Cell Viability Determination

The macrophage cell line J774.1 was grown on sterile Falcon™ 96-welltissue plates (Fisher Scientific, Edmonton) using mMEM without phenolred, supplemented with streptomycin (10 μg/mL), penicillin (10 U/ml),fungizone (0.25 ug/ml) and 5% horse serum. Initial concentration ofmacrophages was about 5000 cells/well. As a control and blank, thebottom row did not have cells. Volumes of 50 μl fresh medium and 50 μlserially diluted (done in triplicate) test sample were added to thewells. After 24 hrs, the media were transferred to a fresh microtitreplate and an equal volume of Griess reagent (1% sulphanilamide, 0.1%napthylethylenediamine dihydrochloride, 2.5% H₂PO₄) was added fornitrate determination (Green et al., 1982). This mixture was incubatedat room temperature for 10 minutes and then read at 540 nm in amicrotitre plate reader (Molecular Devices, Sunnyvale, Calif.). Sodiumnitrate was used as a standard for nitrate quantitation.

Concurrently, cell viability was assessed using AlamarBlue™ (AccuMedInternational Inc., Westlake, Ohio). A tenth ml of a 10% (v/v)concentration was added to each well. The plate was incubated for 5hours at 37° C., 90% humidity, 5% CO₂. The absorbence (570 nm) was readon a Thermomax™ titre plate reader (Molecular Devices, Sunnyvale,Calif.), the bottom row without cells served as the control and blank.

Column Chromatography Fractionation/Purification

Cell lysates (i.e. components released by sonication that went into PBS)and freeze-dried supernatants from F. tularensis LVS cultures werepurified by column chromatography. A twin-pump Pharmacia FPLC™ unit(Amersham Pharmacia Life Sciences, Baie Dorfe, Quebec) was used whichincluded a chart recorder coupled to a ultra-violet detector (214 nm)and a 50 ml loading loop. Two buffers were used. Buffer A comprised of10 mM HEPES, Buffer B comprised of 10 mM HEPES and 3 M KCl. To elutebound components from the column, a gradient was programmed to raise thepercentage of B-buffer to 50% over a time of 20 minutes, following theloading step. To increase binding, samples were dialyzed overnight in aSpectrapor 8 kDa molecular weight cut-off membrane againsttriple-distilled water (to remove salts and other small molecular weightcomponents). The dialyzed material was loaded into the 50 ml loop andsubsequently injected onto the column. The column used was a PharmaciaMono-Q 10/10 column. Flow rate was set at 1.0 ml/minute and the chartspeed was fixed at 5.0 mm/minute. The absorbance at full scale was setat 2.0 with a 10% offset. Fractions were collected in 1.0 minuteintervals.

Results and Discussion (a) Initial Studies

In a previous publication, we reported that by subculturing the F.tularensis live vaccine strain in a synthetic salts medium(Chamberlain's medium) rather than complex peptone-cysteine medium, thatits virulence for mice could be enhanced 1000-fold (Cherwonogrodzky etal., 1994). Our report of a greatly enlarged capsule under these formergrowth conditions “taught away” from subsequent observations of anothervirulence factor. As described in the previous section (see DetailedDescription of the Invention), we had novel undisclosed observationsthat the virulence of F. tularensis LVS (grown in a synthetic medium)was more likely due to a factor other than the capsule. The reasoningfor this was that it appeared that virulence of the bacterium wasunrelated to the presence of capsule (virulence was lost in the third tofifth subculture while capsule formation was unchanged). It was alsoobserved that filter sterilized culture supernatants (free of thebacterial cell) were lethal for mice but that their effect was delayed,causing death only after 1-2 days. Also, the presence of microscopicfoci of necrosis and pneumonia in the lung in infected mice suggestedhost cell death occurred in susceptible lung tissue but not othertissues studied.

The initial work focused on protein profiles of freeze-dried componentssecreted by F. tularensis LVS grown in synthetic medium. The culturesexamined were both agar (i.e. components extracted from the agar andinto liquid PBS) and broths (i.e. the supernatant of broth cultures).Eighty mg of these freeze-dried samples were dissolved in a ml ofdistilled water and used on a 12% SDS-PAGE. The Coomassie stain forproteins was not sensitive enough to detect the low amount of proteinspresent in the samples. A more sensitive silver stain for proteins wasused and indeed proteins were detected with this method.

As we have noted previously in this patent, the pathogenicity of F.tularensis (LVS), subcultured in a synthetic salts medium, appeared tobe transitory. Its ability to kill mice increased until about the thirdsubculture and then diminished after this, even though capsules remainedprevalent throughout these subcultures. FIG. 1 shows that thistransition is also reflected in the silver stain of the supernatants.When the bacterium was subcultured on an agar synthetic salts medium,the protein profiles increased until the third subculture and then thesediminished. For the bacterium subcultured in broth synthetic saltsmedium, only the third subculture showed that proteins were releasedinto the supernatant. The profiles appeared to differ between proteinsreleased when the bacterium was grown in agar medium and proteinsreleased when it was grown in broth medium. It should be noted that asthe bacteria were grown in a simple salts medium, lacking any proteins,all the proteins evident are due to the bacterium and not to any mediumcontaminants.

It is noted that, when one compares the protein profiles of bacterialproteins secreted into the medium of agar or broths, and the proteinprofiles of the cells disrupted by sonication to mimic lysis, thesediffered as shown in FIG. 2. Evidently specific proteins were beingsecreted under these conditions and the profile was not just areflection of all the proteins in the cell that might be released if thecell had lysed.

We interpret the above as a sign of growth stress imposed on thebacterium. It was likely that, during subcultures 1-3, the bacterium wasadapting to the restrictive synthetic salts medium. The stress of beinggrown in a sub-optimal medium may have caused the expression and releaseof components that are also expressed during the course of an infectionwhen the bacterium is acquiring metabolites for growth. Subcultures 4-6may reflect that the bacterium had adapted to the medium, did notrequire additional metabolites and suppressed the expression of thesecomponents.

This same transition of virulence and toxin expression when a bacteriumis grown in synthetic salts medium has also been observed for Vibrioparahaemolyticus and its expression of the Kanagawa hemolysin(Cherwonogrodzky, thesis, 1983).

(b) Characterization of an F. tularensis L VS 52 kDa Protein

The above showed that proteins were present in the supernatants of thedifferent preparation. The results also showed that these and othercomponents were secreted, rather than released by lysis. To date it hasbeen unobvious for an investigator in the field to assess proteinssecreted into the medium from stressed cells grown in a synthetic saltsmedium. Instead, most of the studies deal with membrane proteinsassociated with the bacterial cell grown under optimal conditions.

We investigated the secreted proteins that we had in culturesupernatants to verify whether they have any bearing on the diseaseprocess of tularemia.

A Coomassie Blue or a silver stain shows what proteins are present. Itdoes not necessarily mean that these proteins secreted into the mediumare significant in the disease process. To resolve this, the mediumextracts or the broth supernatants were freeze-dried and theninvestigated with Western immunoblots (antigens are separated byelectrophoresis, transferred to a membrane, and the antibodies in serumfrom affected animals are used as probes to identify antigens ofimportance in the disease process). Initially a commercial rabbitanti-tularemia antiserum was used. Results were negative (data notshown). The antibodies produced in rabbits that were vaccinated withkilled F. tularensis cells (the SCHU strain grown in complex medium) didnot recognize the antigens released into the medium that we havediscovered with a live culture of F. tularensis LVS grown in a syntheticsalts medium.

Next, antisera from mice that had been infected with B. abortus wereused. As B. abortus and F. tularensis proteins and O-polysaccharide(OPS) cross-react (Van Hoek et al., manuscript in preparation, dataunpublished as yet) it was thought that mouse anti-Brucella serum mightserve as an antibody source to identify F. tularensis antigens. Resultswere again negative (data not shown, negative results on DRES file98-08-04-b).

It was then undertaken to prepare antisera unobvious to one skilled inthe art of tularemia. At our DRDC Suffield laboratory, we have developedan O-polysaccharide vaccine from B. abortus that protects mice frombrucellosis. This vaccine also cross-protects mice from tularemia(results unpublished). We vaccinated mice with the OPS vaccine againstB. abortus and then infected these animals with several lethal doses (10LD₅₀) of F. tularensis LVS. In the past usually no unvaccinated controlmice survived this dose. In contrast, usually 60% of vaccinated micewill survive this multiple lethal dose infection and indeed during thisstudy 12 of 20 vaccinated mice survived. The antisera from mice that hadsurvived the course of tularemia would likely have antibodies toantigens related to the disease process caused by the living bacterium.Upon doing immunoblots of cell lysates with this antisera (from micethat survived tularemia), antibodies did label components not evidentwith antisera from rabbits vaccinated with killed whole cells of F.tularensis nor mice infected with B. abortus. The predominant proteindetected was estimated to have a molecular weight around 52 kDa (seeFIG. 3).

When supernatant preparations that contained this 52 kDa protein weredigested with proteinase K, it vanished, showing that it was indeed aprotein (DRES file 00-03-13-a). Also, we observed thatpost-translational modifications did occur but that these were notconsistent. On some occasions this protein appeared to be a doublet of52 and also 53 kDa (DRES file 98-07-27-a). Usually the 52 kDa proteindid not have carbohydrate as evidenced by a lack of reaction withperiodate treatment followed by silver staining. On the occasions whenit did show glycosylation (unfiled, photograph available) tentatively wecan describe this as being similar to the O-polysaccharide of B. abortusbecause the anti-52 kDa antiserum also reacted with a component in celllysates of Escherichia coli O: 157H:7 and Pseudomonas maltophilia 555.The only component known to be common to B. abortus, F. tularensis andthese latter two bacteria is the O-polysaccharide (data not shown, DRESphotograph file number 00-04-17-a).

It was also observed that the immunoserum bound to the high molecularweight protein myosin that was in the molecular weight proteinstandards. We did not pursue whether this binding was due tocross-reaction between myosin and F. tularensis proteins, or whether asa consequence of active tularemia the mice had made auto-antibodies totheir own myosin. In either case, anti-myosin antibodies might be anovel indirect test for identifying tularemia in an animal or human.

It was investigated whether this 52 kDa protein, initially found outsidethe cell grown in agar or broth synthetic salts medium, could also befound inside the cell when the above mentioned immunoserum was used.Usually the third broth subculture yielded the greatest amount of 52 kDaprotein in the supernatant. Francisella tularensis was subcultured twicein broth, and then 1 ml was transferred to eight 500 ml flaskscontaining 100 ml of synthetic salts medium. Every day afterwards, aflask was removed, the culture was centrifuged (10,000×g, 30 minutes, 4C), the supernatant discarded, the cells resuspended in 10 ml of saline,the cells were disrupted by sonication, and then the preparations werestandardized by diluting to 3 mg/ml. FIG. 4 shows that the 52 kDaprotein is also found within the cell, although it appears to increaseuntil the culture reaches stationary phase (i.e. day 5, note that itappears as a doublet). Afterwards it appears to persist though it doesappear to decrease in amounts as the culture matures (i.e. until day 8).

(c) Identification of the 52 kDa Protein as a Toxic Virulence Factor

Other researchers have studied the proteins of F. tularensis LVS, buttheir investigations led to the study of low molecular weight proteins,specifically a 17 kDa protein (Sjostedt, 1997) that did not appear to bea virulence factor. In our study, a striking result of immunostainingwith the use of antiserum, taken from vaccinated mice that survivedmultiple lethal doses of F. tularensis LVS, was the identification of aprominent F. tularensis 52,000 molecular weight (52 kDa) protein.Obviously it played some role in the disease process. We verifiedwhether this 52 kDa protein was, in fact, a toxic virulence factor of F.tularensis.

The 52 kDa protein was purified by taking the supernatant of the thirdsubculture of F. tularensis LVS grown in the synthetic salts medium(removing the cells by centrifugation), filter sterilizing this througha 0.45 μm filter, fractionating and concentrating the solution by usingan Amicon™ filtration with 30,000 m.w. cutoff (i.e. proteins greaterthan 30 kDa were retained). This concentrate was then fractioned on aPharmacia Mono-Q™ column (which separates proteins on the basis ofcharge and hence isoelectric points, see FIG. 5). Proteins were elutedwith increasing concentrations of potassium chloride. Dialysis againstsaline removed this potassium chloride. Although Fraction 24 had thegreatest amount of the 52 kDa protein (as shown by the samples beingelectrophoresed on SDS-PAGE gels, followed by Coomassie Blue™ staining),Fraction 29 was used in further studies because despite being moredilute, contaminating proteins were less evident.

When HELA (human, cancer) cell cultures were first tested forsensitivity to this 52 kDa protein, no effect was observed. However,when Vero (Green Monkey Kidney Cells) cell cultures were used, celldeath was observed, especially if the preparation of the 52 kDa proteinwas concentrated by freeze-drying and then dissolved in a minimal amountof sterile saline. The 52 kDa protein also affected cell cultures ofJ774A.1 cells (derived from balb/c mouse macrophages). The test samplewas standardized by the amount of protein given. When the amount ofprotein was greater than 0.5 mg/ml, cell death for both tissue culturesdid occur (about 50% death at 1 mg/ml) (FIG. 6) but only after 24 hrincubation. These results support the preliminary observations made adecade previously that F. tularensis produces a component that is toxicbut that its effects are not immediate and it affects only susceptibletissues. For protein amounts less than 0.1 mg/ml, cell death was notobserved.

When mammalian cells are stressed, these may produce nitrous oxide. ForJ774A.1 mouse macrophage cells, nitrous oxide production correlated withthe amount of the 52 kDa protein in the different Pharmacia Mono-Q™fractions noted above (FIG. 7). Nitrous oxide also increased whenprotein amounts went from the sublethal amounts of 0.1 mg/ml to 0.5mg/ml (FIG. 8). For higher amounts, the cells had been killed and hencedid not express nitrous oxide. With the 52 kDa protein stressing themammalian cells to produce nitrous oxide, or this protein killing thecells at higher concentrations, our results strongly suggest that the 52kDa protein was a toxin. The observation a decade ago of delayed deathfor mice given 0.050 ml of culture supernatant intranasally can now bedescribed as initiation of host cell stress and a cascade of negativemetabolic responses.

(d) Vaccine/Immunization Studies

Experiments were conducted to determine whether this 52 kDa protein wasa potential vaccine candidate.

As time restraints allowed only a brief inspection of vaccine potential,a rapid screen was done. Two hundred and fifty (250), rather than 10,LD₅₀s of F. tularensis LVS were given to eliminate components of onlymarginal vaccine efficacy. The infection was given intranasally, ratherthan intraperitoneal, to reflect a biological warfare scenario ofchallenge. Outbred CD1 mice, rather than inbred balb/c mice, were usedto reflect genetic diversity more relevant to human populations. Micewere monitored for 3 weeks rather than 1 week to account for any delayedresponses and to ensure that recovery was complete. Table 1 shows thatsome of these vaccine, or vaccine combinations, gave obvious protectioneven under these exceptionally harsh challenges.

TABLE 1 Assessment of different vaccine candidates for the protection ofCD1 mice against 250 LD₅₀s of F. tularensis LVS given intranasally.PROTECTION FROM CHALLENGE (from 250 LD₅₀ GROUP VACCINATION (given i.p.)F. tularensis LVS) 1 Control 0/5 mice (0%) (no vaccination, only 0.1 mlsterile PBS/mouse given) Different Brucella vaccine formulations: 2 B.abortus 1119-3 O-polysaccharide (OPS) (1 μg/mouse) 2/5 mice (40%) 3 B.melitensis 16M OPS (1 μg/mouse) 0/5 mice (0%) 4 B. suis 145 OPS (1μg/mouse) 0/5 mice (0%) F. tularensis cell lysate 5 F. tularensis celllysate (2 μg/mouse) 5/5 mice (100%) F. tularensis broth culturesupernatant, fractioned by Amicon 6 > (Greater than) 30 kDa (2 μg/mouse)1/5 mice (20%) 7 >30 kDa (2 μg/mouse) + B. suis OPS vaccine (1 μg/mouse)5/5 mice (100%) Purified 52 kDa component (Fraction #29) 8 Fraction #29(2 μg/mouse) 1/5 mice (20%) 9 Fraction #29 (2 μg/mouse) + B. suis OPSvaccine (1 μg/mouse) 2/5 mice (40%)

From the above, all control mice (Table 1, Group 1) died from this highdose of F. tularensis LVS as expected.

Different species of Brucella will express different forms of thepolysaccharide on the cell wall. When different BrucellaO-polysaccharide vaccines were tested, B. abortus OPS (Group 2) appearedto give the best protection against tularemia in mice. It is likely thatthe other polysaccharides are also protective, but only against a lowerlethal dose than that used in our screening experiments. We have foundthat the polysaccharide of Brucella abortus cross-reacts with thepolysaccharide of F. tularensis LVS (unpublished data). The significanceof these sugars, and glycosylation of proteins, will be discussed below.

In the past, vaccination with whole killed F. tularensis cells have notgiven immunity for mice. This teaches away from our own results where weused F. tularensis cells, stressed in a synthetic salts medium toexpress the 52 kDa protein, then lysed by sonication to release thiscomponent (unbroken cells and cellular debris were removed bycentrifugation). Using this noted lysate gave remarkable totalprotection for the mice from tularemia (Group 5).

The entire bacterial lysate may be unnecessary to protect mice fromtularemia. A fraction of proteins greater than 30,000 molecular weightwas prepared with the use of the Amicon™ filtration unit. Although someprotection was noticed for this fraction (Group 6), remarkable totalprotection occurred when bacterial polysaccharide was included (Group7).

As our studies have shown that the 52 kDa protein was identified as asignificant factor in the disease process of tularemia (i.e. byantibodies in “survivor” serum), we vaccinated mice with this component(Group 8). It did protect to a minor extent and this protection wasenhanced with B. suis OPS vaccine (Group 9). In hindsight, we shouldhave vaccinated the mice with polysaccharide from either B. abortus orfrom F. tularensis LVS to enhance the efficacy of the 52 kDa protein.

As noted previously, for immunoblots with lysates of Escherichia coliO:157, H:7, Salmonella godesberg, and Pseudomonas maltophilia 555,antibodies (from the serum from vaccinated mice that survived tularemia)strongly bound to some of the proteins. As these bacteria sharecross-reactive polysaccharides, the results suggest thatcross-protection was due in part to cross-reaction to the carbohydrates.As noted previously in this patent, we did find that the 52 kDa proteinwas glycosylated in some cultures of F. tularensis LVS. We believe thatthis glycosylation enhances the entry of this toxin into its targetcells. One can either protect a mammal from the effects of the toxin byvaccinating with the toxin (Group 8) and by logical extension a toxoid,with the glycosylating component or a polysaccharide similar to thiscomponent (Group 2) or with both the toxin and the polysaccharide whichact synergistically to enhance protection (Group 7 and Group 9).

(e) Additional Studies

Time allowed us only to pursue the 52 kDa protein, which appeared to bethe most striking of proteins secreted or released by F. tularensis LVSstressed in a synthetic salts medium. However, there were other proteinsof note (see FIG. 9). When the preparation was digested with proteinaseK, these proteins were digested and did not appear in polyacrylamidegels (Lane 3). Digestion with lipase caused the loss of a 45 kDalipoprotein (lane 5). Digestion with DNAse caused the shift of a 19 kDanucleoprotein to a higher 33 kDa protein, suggesting dimerization (Lane7). Aside from identification by these preliminary characterizations,time did not allow us to assess their vaccine potential or whether thesecould act synergistically with the 52 kDa protein.

In the model of diphtheria toxin, bacteriophage influence the expressionof toxins in Corynebacterium diphtheriae. We investigated if F.tularensis LVS harboured a bacteriophage that, although usuallyrepressed and lysogenic with the bacterial chromosome, under thestressful condition of the bacterium being cultured in a syntheticmedium, emerged to lyse the bacterium and hence release cell associatedcomponents such as the 52 kDa protein. We attempted to isolate phage,plasmids and extra-chromosomal material from culture cells andsupernatants but were not successful.

It is to be understood that the embodiments and variations shown anddescribed herein are merely illustrative of the principles of thisinvention and that various modifications may be implemented by thoseskilled in the art without departing from the scope and spirit of theinvention.

1. A method of assessing the immune status and level of protection for amammal vaccinated with an isolated subcellular protein having amolecular weight of about 52 kDa, said protein having been expressedfrom a Francisella tularensis subculture growing in synthetic saltsmedium at pH 6.5, comprising detecting the presence of antibodies tosaid protein in said mammal.
 2. A method for assessing in vitro theusefulness of a vaccine lot for quality assurance, comprisingidentifying and quantifying an isolated subcellular protein having amolecular weight of about 52 kDa, said protein having been expressedfrom a Francisella tularensis subculture growing in synthetic saltsmedium at pH 6.5, in said lot.
 3. The method of claim 2, wherein saidvaccine lot is a Francisella tularensis vaccine lot.